Tcs sp5 aobs inverted confocal microscope

Fresh cultures at OD660∼0.4 were washed twice using mM63 medium, and then resuspended in mM63 medium while adjusting the OD660nm = 0.15 per bacterial strain, in order to adjust the initial cell density for experiments. For range expansions in environments including ampiciline, we used an initial OD660nm = 0.3 per bacterial strain. 0.4 μL of the corresponding cultures where then inoculated in mM63 1.2% agar plates (supplemented with amino acid and antibiotic as required by the experimental scenario). Colonies were incubated for 4 days (7 days for the case of front speed measurements) at 37°C and humidity 90%.
Colonies were observed using a Leica TCS SP5 AOBS (inverted) confocal microscope.

SlmA, SBScons and FtsZ containing GUVs made of egg-yolk L-α-phosphatidylcholine (EPC, Avanti Polar Lipids, Alabaster, AL) were prepared according to a water-in-oil double emulsion method recently optimized [42 (link)]. Vesicles were made permeable by mixing, prior to lipid film formation, EPC with 10 mol% of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, Avanti Polar Lipids, Alabaster, AL) that induces heterogeneities in the vesicle membrane at temperatures above its Tm (23°C [43 (link)]) through which small ligands can diffuse. External addition of MgCl₂ (3–4 mM) and GTP (2 mM) triggered the polymerization of FtsZ (12 μM) into bundles inside the GUVs. The protein was equilibrated in 50 mM Tris-HCl pH 7.5 containing 300 mM KCl, 100 mM sucrose and 50 g/L Ficoll 70. When present, 1–50 μM SlmA, 0.2–2 μM SBScons and 1.2 μM FtsZ-Alexa 488 were added to this solution. Visualization of precipitated GUVs was conducted on a Leica TCS SP5 AOBS inverted confocal microscope (Leica, Mannheim, Germany) as previously described [42 (link)].

Immunofluorescence analyses were performed to investigate osteogenic and adipogenic differentiation or autophagy in in vitro experiments. After 21-day culture, cells were fixed and processed according to the manufacturer's protocols. Primary antibodies were diluted (as reported in the datasheet) in antibody dilution buffer and incubated overnight at 4°C. To evaluate osteogenic differentiation and autophagy, we used osteocalcin C8 antibody (sc74495, Santa Cruz, Dallas, Texas, USA) and LC3B (cat. #2775, Cell Signaling, Leiden, The Netherlands), respectively. Assessment of adipogenic stimuli was achieved by testing perilipin D1D8 (cat. #12589, Cell Signaling). Slides were then incubated with secondary antibodies Alexa Fluor® 594 anti-rabbit (cat. #r37117), Alexa Fluor® 488 anti-rabbit (cat. #a-11034), and goat mouse fluorescein conjugated (cat. Ap124f, Millipore, Burlington, Massachusetts, USA). Nuclear staining was performed by ProLong™ Gold Antifade Mountant with DAPI. Images were recorded using a Leica (Wetzlar, Germany) TCS SP5 AOBS inverted confocal microscope at 63x. To express data in a semiquantitative way, four different fields were measured for each sample, in three independent experiments with about 80–100 total cells.